Simultaneous Alternating and Direct Current Readout of Protein Ion Channel Blocking Events Using Glass Nanopore Membranes

• Published in: Analytical chemistry (Washington) 2008-03, Vol.80 (6), p.2069-2076
• Main Authors: Ervin, Eric N, Kawano, Ryuji, White, Ryan J, White, Henry S
• Format: Article
• Language: English
• Subjects: Analytical biochemistry: general aspects, technics, instrumentation; Analytical chemistry; Analytical, structural and metabolic biochemistry; Base Sequence; Biochemistry; Biological and medical sciences; DNA, Single-Stranded; Fundamental and applied biological sciences. Psychology; Glass; Ions; Kinetics; Membranes; Membranes, Artificial; Nanotechnology; Proteins; Proteins - chemistry
• ISSN: 0003-2700; 1520-6882
• DOI: 10.1021/ac7021103

Alternating current (ac) phase-sensitive detection is used to measure the conductance of the ion channel α-hemolysin (αHL), while simultaneously applying a direct current (dc) bias to electrostatically control the binding affinity and kinetics of charged molecules within the protein lumen. Ion channel conductance was recorded while applying a 10−20 mV rms, 1−2 kHz bias across a single αHL protein inserted in a 1,2-diphytanoyl-sn-glycero-3-phosphocholine lipid bilayer that is suspended across the orifice (100−500 nm radius) of a glass nanopore membrane. Step changes in the ac ion channel conductance with a temporal response (t 10 - 90) of 1.5 ms and noise amplitude of ∼2 pA were obtained using a low-noise potentiostat and a lock-in amplifier. These conditions were used to monitor the reversible and stochastic binding of heptakis-(6-O-sulfo)-β-cyclodextrin and a nine base pair DNA hairpin molecule to the ion channel. Alternating current methodology allows the binding kinetics and affinity between the protein ion channel and analyte to be investigated as a function of the dc bias, including ion channel conductance measurements in the absence of a dc bias.